P339: DEVELOPMENT OF ANTI-IL-7RΑ ANTIBODIES AS A TARGETED THERAPY FOR T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: Mafalda Duque, João Santos, Sílvia Andrade, Daniela Teixeira, Maria Pajuelo, Rita Fragoso, João T. Barata
Journal: HemaSphere (2023)

Background: T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive sub-type of ALL that accounts for approximately 15% of pediatric and 25% of adult ALL cases. Despite the significant improvements in treatment outcome of pediatric patients, the prognosis of adult patients is still poor. Furthermore, the lack of specificity of current therapies is associated with dismal prognosis for relapsed patients, and with debilitating side-effects. Interleukin-7 (IL-7) and its receptor (IL-7R) have been implicated in T-ALL development and maintenance. IL-7Rα is expressed in around 70% of T-ALL cases and IL-7 is known to promote T-ALL cell proliferation and viability in vitro and leukemogenesis in vivo. Furthermore, 10% of T-ALL cases display IL-7Rα gain-of-function mutations that lead to constitutive activation of downstream signaling. Aims: The dependence of T-ALL cells on IL-7Rα provides a robust rationale to target this pathway for therapeutic purposes, which we are exploring by functionally characterizing novel anti-IL-7Rα monoclonal Antibodies (mAbs). Methods: To identify anti-human IL-7Rα mAbs with clinical potential, we have evaluated the ability and specificity of a panel of 48 mAbs (generated by phage display by FairJourney Biologics) to bind to human IL-7Rα, by titration of each Ab on T-ALL cell lines, using flow cytometry (FC). mAbs with high binding capacity were further characterized for: 1) ability to functionally block IL-7-mediated signaling on T-ALL primary cells and cell lines (western blot with phospho-specific antibodies); 2) impact on the viability and proliferation of T-ALL primary and patient-derived xenograft (PDX) samples (FC analysis); 3) internalization kinetics (FC analysis); and 4) capacity to induce Natural Killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Results: Four mAbs with high affinity binding were selected for functional characterization (FJB26, FJB29, FJB45 and FJB48). None of these mAbs blocked IL-7Rα-mediated signaling in T-ALL cell lines and PDX cells, as evidenced by the lack of a significant impact on the phosphorylation levels of the main downstream effectors of IL-7R (STAT5, AKT, ERK and S6) and on IL-7-mediated viability and proliferation of T-ALL cells. However, they all showed ADCC potential. FBJ45 displayed the strongest effect, inducing cytotoxicity levels of up to 98% in T-ALL PDX cells. Interestingly, FBJ48, which displayed lower ADCC potential (~15%), had a high internalization rate in T-ALL cells (50% of IL-7Rα internalization after 30 minutes of Ab exposure). Summary/Conclusion: Of the 48 mAbs initially screened for high affinity, we selected four Abs for functional in vitro analysis. Of these, at least one mAb (FBJ45) displayed high capacity to promote T-ALL ADCC. We are in the process of validating this mAb in vivo using T-ALL xenograft mouse models. We have further identified FBJ48 as having good internalization kinetics, which we will further characterize to determine its potential for antibody-drug conjugate (ADC) development. Keywords: Antibody, IL-7, T-ALL, Targeted therapy